Insulin-like growth factors (IGFs) are part of a complex system that cells use to communicate with their physiologic environment. This complex system (often referred to as the insulin-like growth factor axis) consists of two cell-surface receptors (IGF-1R and IGF-2R), two ligands (IGF-1 and IGF-2), a family of six high-affinity IGF-binding proteins (IGFBP 1-6), and associated IGFBP degrading enzymes (proteases). This system is important not only for the regulation of normal physiology but also for a number of pathological states (Glass, Nat Cell Biol 5:87-90, 2003).
The IGF axis has been shown to play roles in the promotion of cell proliferation and the inhibition of cell death (apoptosis). IGF-1 is mainly secreted by the liver as a result of stimulation by human growth hormone (hGH). Almost every cell in the human body is affected by IGF-1, especially cells in muscles, cartilage, bones, liver, kidney, nerves, skin and lungs. In addition to the insulin-like effects, IGF-1 can also regulate cell growth. IGF-1 and IGF-2 are regulated by a family of gene products known as the IGF-binding proteins. These proteins help to modulate IGF action in complex ways that involve both inhibiting IGF action by preventing binding to the IGF receptors as well as promoting IGF action through aiding delivery to the receptors and increasing IGF half-life in the blood stream. There are at least six characterized binding proteins (IGFBP1-6).
In its mature form, human IGF-1, also called somatomedin, is a small protein of 70 amino acids that has been shown to stimulate growth of a wide range of cells in culture. The IGF-1 protein is initially encoded by three known splice variant mRNAs. The open reading frame of each mRNA encodes a precursor protein containing the 70 amino acid IGF-1 (SEQ ID NO.:1) and a particular E-peptide at the C-terminus, depending on the particular IGF-1 mRNA. These E-peptides have been termed the Ea (rsvraqrhtdmpktqkevhlknasrgsagnknyrm; SEQ ID NO.:2), Eb (rsvraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkk gk; SEQ ID NO.:3) and Ec (rsvraqrhtdmpktqkyqppstnkntksqrrkgstfeerk; SEQ ID NO.: 4) peptides and range from 35 to 87 amino acids in length and encompass a common sequence region at the N-terminus and a variable sequence region at the C-terminus. For example, the wild-type open reading frame for the IGF-1-Ea encodes a polypeptide of 135 amino acids including the leader sequence and a polypeptide of 105 amino acids without the leader sequence (gpetlcgaelvdalqfvcgdrgfyfnkptgygsssrrapqtgivdeccfrscdlrrlemycaplkpaksarsvraqrh tdmpktqkevhlknasrgsagnknyrm; SEQ ID NO.:5). In physiological expression, the E-peptides are cleaved off of the precursor by endogenous proteases to yield the mature 70 amino acid IGF-1. The availability and half-life of IGF-1 in human serum is mainly influenced and modulated by proteases and IGF-1 binding proteins (IGFBP's). IGFBP's can either inhibit or potentiate IGF-1 activities (Oh Y, et al., Characterization of the affinities of insulin-like growth factor (IGF)-binding proteins 1-4 for IGF-I, IGF-II, IGF-I/insulin hybrid, and IGF-I analogs. Endocrinology. 1993 March; 132(3):1337-44). Strategies to increase the half-life of IGF-1 have been described in the prior art. Strategies that have been contemplated are
(i) the production of IGF-1 variants comprising specific mutations aiming to prevent the cleavage of IGF-1 in human serum by serine proteases, or to alleviate the negative impact of IGF-1 binding proteins on the availability or serum half-life of IGF-1 (WO200040613, WO05033134, WO2006074390, WO2007/146689);(ii) the production of IGF-1 fusion proteins, wherein the mature IGF-1 protein is fused to a human immunoglobulin Fc region (WO2005033134, WO200040613);(iii) the use of IGF-1 precursor proteins wherein cleavage of the E-peptide from IGF-1 by a protease is reduced by modification of the precursor protein (WO2007146689);(iv) combinations of the above described strategies ((i)/(ii) WO05033134, (i)/(ii) WO200040613, (i)/(iii) WO2007146689).
Despite the above described strategies, the IGF-1 precursor variants fused to a human immunoglobulin Fc region remain poor drug candidates for mainly two reasons: (i) low production yield in mammalian production system, and (ii) increased binding affinity to the insulin receptor (InsR) compared to the unmodified human wildtype IGF-1, which can result in hypoglycemia, an adverse event of therapeutic concern.
Consequently, there exists a need for a technology which overcomes the above described prior art problems. The present invention addresses this need in a number of aspects.